Direct gene transfer via cationic lipid/plasmid DNA (pDNA) complexes offers an attractive therapeutic approach to cystic fibrosis. The objective of this study was to examine the safety and efficacy of aerosol delivery of up to 12 doses of lipid/pDNA into the lungs of nonhuman primates. The time interval between doses was r 7 days. Aerosol exposures prior to the last exposure were to a mid-level dose of lipid/pDNA with a plasmid carrying the gene for the cystic fibrosis transmembrance conductance regulator (CFTR). The last aerosol exposure was to the same dose of lipid/pDNA with a plasmid carrying the gene for chloramphenicol acetyltransferase (CAT). Rhesus monkeys (Macaca mulatta) were trained by the New England Regional Primate Research Center personnel to sit in a standard primate restraint chair and to wear a fitted face mask through which aerosolized lipid/pDNA was delivered for up to 3 hours. Prior to aerosol administrations thoracic radiographs, serum chemistries, CBCs and antibody to lipid and or DNA was determined. During aerosol administrations the animal's heart rate, blood oxygen saturation and clinical condition was monitored. At predetermined intervals during aerosol treatments animals were anesthetized and thoracic radiographs, nasal brushings, bronchoscope guided bronchial brushes were obtained. Serum chemistries and CBCs were also performed. Bronchial and nasal brushes were analyzed for pDNA specific DNA and mRNA by fluorescent in situ hybridization (FISH) and RT-PCR, respectively. Two days following the last lipid/pDNA exposure thoracic radiographs, a serum chemistry, CBC and serum for detection of any antibodies to lipid or pDNA were obtained. The animal was euthanized and underwent a complete necropsy. The lungs were evaluated for transgene expression, and pDNA specific mRNA and DNA. The lungs, livers, kidneys, spleens, hearts and any other organs displaying abnormalities on gross examination were examined histologically. The first animal received 12 aerosol administrations of lipid/pDNA with one week between each dose. The second animal received 6 aerosol administrations of lipid/pDNA with an interval of s 2 months between doses. Throughout aerosol exposures and just prior to euthanasia thoracic radiographs, serum chemistries and CBCs were within normal limits. Analysis of transgene expression indicated that a longer interval between exposures resulted in higher levels of transgene expression. Vector DNA was detected in nasal and bronchial brushes. Transgene expression was detected in lung but not on brushes, suggesting that the lack of transgene expression on brushes was a sampling artifact. In the first animal there was minimal inflammation in the lung and all other organs